The present invention relates to a liquid chromatograph such as a high-performance liquid chromatograph (HPLC). In particular, the present invention relates to a two-dimensional liquid chromatograph in which a first-dimension analysis column separates components and a trap column retains the components, and a second-dimension analysis column separates the components one more time.
A liquid chromatography includes various separation modes such as a normal phase, a reversed-phase, ion exchange, and size exclusion. A one-dimensional liquid chromatography uses one of the separation modes. It is sometimes difficult to analyze a sample with the one-dimensional liquid chromatography depending on a sample. For example, in a field of proteome analysis in which a biological sample is analyzed, when high-performance liquid chromatography is used to analyze components in one separation mode, a large number of eluted components are present, thereby overlapping peaks of the components. Accordingly, even when a mass spectrometer with high resolution is used as a detector, it is difficult to perform the analysis.
In order to solve the problem, a two-dimensional liquid chromatography capable of combining two separation modes with different mechanisms has been used. In the two-dimensional liquid chromatography, generally an ion exchange mode is used for separation according to ion strength as first-dimension separation, and components eluted from the ion exchange column are analyzed with a reversed-phase mode as second-dimension separation.
More specifically, an ion exchange column for the first-dimension separation and a reversed-phase column for the second-dimension separation are connected in series, and a detector is connected at a downstream side thereof. In the first-dimension separation, a mobile phase of the ion exchange mode flows in the ion exchange column for separating the components through a concentration gradient according to ion strength. In this case, only a part of the components is eluted and guided to the reversed-phase column. Then, the mobile phase is switched to the reversed-phase, and the part of the components eluted in the first-dimension is analyzed in the reversed-phase column while desalting as the second-dimension analysis to be detected by the detector. After the second-dimension separation and analysis, the mobile phase is switched again to the ion exchange mode to change the ion strength, so that the next eluted components are eluted through the first-dimension column. The mobile phase is switched again to the reversed-phase mode, and the desalting and second-dimension separation and analysis are performed.
Through the process, the two-dimensional liquid chromatography is performed by repeating the first-dimension separation and second-dimension separation. In the two-dimensional liquid chromatography, it takes a long period of time to replace the mobile phases in the system, thereby making it difficult to efficiently perform the analysis.
In order to solve the problem, a trap column has been provided. In this system, a first-dimension separation channel and a second-dimension separation channel are provided separately. After a component eluted from the first-dimension column is retained in the trap column, the channels are switched, and the component captured in the trap column is separated and analyzed by the second-dimension separation channel.
As compared with the system having two columns connected in series for the first-dimension and second-dimension separations, this system has an advantage in which a mobile phase liquid delivery pump for the first-dimension analysis in the ion exchange mode and a mobile phase liquid delivery pump for the second-dimension analysis in the reverse mode are provided separately, and the trap column is provided. When a part of the components is eluted at a certain concentration of ion strength and retained in the trap column in the first-dimension separation, the first-dimension mobile phase liquid delivery pump is stopped. Then, the channel switching valve connected to the trap column is switched, so that the second-dimension analysis is performed. When the second-dimension analysis is finished, the first-dimension pump is started again. Then, the concentration is changed, and the next eluted components are retained in the trap column. Then, the first-dimension pump is stopped, and the channel switching valve is switched, so that the second-dimension analysis is performed. By repeating the steps, the two-dimensional liquid chromatography is performed.
The system has an advantage in which the liquid delivery pumps for the respective mobile phases are separately provided, and the second-dimension analysis is performed in a state in which the component eluted in the first-dimension separation is concentrated. The system has a disadvantage in which it is necessary to stop the first-dimension liquid feed pump several times during the analysis, thereby lowering reproducibility. Further, the first-dimension analysis channel of the channel switching valve becomes an open system when the first-dimension separation is switched to the second-dimension analysis. Accordingly, the first-dimension mobile phase may leak from the first-dimension channel due to a residual internal pressure in the channel, thereby losing a component.
In order to solve the problem, a plurality of trap columns has been proposed (see Patent Document 1). In this system, all of the components are separated and eluted while applying a concentration gradient in the first-dimension separation, and all of the eluted components are retained by the plural trap columns. Then, the trap columns are successively connected to a channel for the second-dimension separation, thereby performing the second-dimension analysis. The system has an advantage in which the first-dimension separation is not interrupted during the analysis, thereby improving reproducibility of the analysis is good and eliminating leak of the components.
Patent Document 1: Japanese Patent Publication (Kokai) No. 2003-254955
In the method using the plural trap columns for retaining the eluted components separated in the first-dimension separation, it is possible to fractionate the components eluted in the first-dimension separation into only the number of the trap columns. However, when a sample contains a large number of components, it is necessary to provide a large number of trap columns, thereby increasing a size of an apparatus.
In view of the problems described above, an object of the present invention is to provide a two-dimensional liquid chromatograph capable of analyzing a large number of components with a minimum number of trap columns.
Further objects and advantages of the invention will be apparent from the following description of the invention.